Effects of the whz mutation on embryonic heart morphology and growth.

(A-D) Lateral view of wild-type (wt; A, C) and whz mutant (B, D) embryos at 72 hours post fertilization (hpf). Whz mutants show pericardial edema, blood congestion at the cardiac inflow tract and stretched heart chambers. (E, F) Hematoxylin and Eosin staining of sagittal histological sections of wt (E) and whz mutant (F) hearts at 72 hpf. Similar to wild-types, in whz atria and ventricles, myocardial (myo) and endocardial (endo) cell layers are clearly defined and separated by an atrio-ventricular canal (AVC). In contrast to wild-type hearts, whz mutant ventricles appear small and the myocardium monolayered. (G-I) Dissected wt (G) and whz mutant (H) hearts at 72 hpf, stained with a cardiomyocyte-specific MEF-2 antibody (nuclei; red) and co-stained with S46, exclusively marking atrial cardiomyocytes (green). (I) whz mutant hearts show significantly reduced ventricular cardiomyocytes at 72 hpf (sib: 144.2±10 SD and whz: 94.9±10 SD, n = 10; p<0.0001), whereas cardiomyocyte numbers are comparable between wt and whz ventricles at 48 hpf (wt: 93.4±10 SD and whz: 88.2±10 SD, n = 10; p>0.05).

The whz mutation interferes with cardiomyocyte proliferation.

(A, B) TUNEL stainings of embryonic zebrafish hearts at 72 hpf show no difference in the number of apoptotic cardiomyocytes in wt (A) and whz (B) mutant embryos. TUNEL positive cells in the pericardium (peri) and ventricles are marked by arrows. (C-F) Dissected wt (C) and whz mutant (D) hearts at 72 hpf, stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize cardiomyocyte proliferation. At 48 hpf, proliferation of ventricular cardiomyocytes appears unaltered between wt and whz mutant hearts (sib: 5±2% SD and whz: 4±2% SD, n = 10; p>0.05) (E), whereas cardiomyocyte proliferation in whz mutant ventricles is significantly reduced compared to wt at 72 hpf (sib: 8±2% SD, whz: 2±2% SD, n = 10, p = 0.0001) (F).

whz encodes Tbox transcription factor 20 (tbx20).

(A) Integrated genetic and physical map of the whz locus on zebrafish chromosome 16. The whz mutation interval is flanked by the microsatellite markers z1215 and z6240 and encodes 2 open reading frames, zebrafish tbx20 and herpud2. (B) Missense mutation of Adenine to Cytosine of the stop codon of tbx20 results in the loss of the original stop codon and the termination of tbx20 transcription after 87 additional nucleotides. (C) Partial amino acid alignment of the C-terminus of zebrafish whz and wt as well as human and murine Tbx20. Tbx20 is highly conserved cross-species and the whz mutant Tbx20 protein is extended by 29 additional amino acids. (D, E) Tbx20-specific whole-mount antisense RNA in situ hybridization detects unaltered expression of zebrafish tbx20 in whz mutant embryos compared to wild-types. (F) Quantitative RT-PCR analysis showing similar relative mRNA levels of tbx20 in wt and whz embryos at 72 hpf (n = 4; p = 0.5957). (G, H) Tbx20 protein levels are significantly reduced in whz mutant embryos compared to wild-type littermates (n = 4; p = 0.0286).

Knock-down of zebrafish tbx20 phenocopies the whz mutant phenotype.

(A-D) Lateral view of wild-type embryos injected with zebrafish tbx20-specific control Morpholinos (MO-ctrl) (A, C) and tbx20 start and splice Morpholinos (MO-tbx20) (B, D) at 72 hpf, respectively. Knock-down of tbx20 phenocopies the whz mutant phenotype, whereas injection of the same amount of specific-control Morpholinos does not affect heart growth. (E, F) Hematoxylin and Eosin staining of sagittal histological sections of MO-ctrl (E) and MO-tbx20 (F) injected hearts at 72 hpf. In contrast to control hearts, tbx20 morphant ventricles appear small and the myocardium monolayered. (G) 78% (MO1-tbx20) and 76% (MO2-tbx20) of the injected embryos are indistinguishable from whz mutant embryos. (H) Tbx20 morphant hearts show significantly reduced ventricular cardiomyocytes at 72 hpf (MO1-control: 130.7±10 SD, MO1-tbx20: 86.5±10 SD, n = 10; p = 0.0001). (I-K) Cardiomyocyte proliferation in Tbx20 morphant ventricles is significantly reduced compared to controls at 72 hpf (MO1-control: 6±2% SD, MO1-tbx20: 1±2% SD, n = 10; p = 0.0001).

Ectopic expression of wild-type Tbx20 rescues whz mutant embryos.

(A, B) Lateral view of whz mutant embryos control-injected with KCl (A) and wild-type zebrafish tbx20 mRNA (B), respectively. (C) Ectopic expression of wild-type zebrafish tbx20 mRNA can rescue the heart phenotype of 73% of homozygous whz mutant embryos, whereas injection of KCl has no effect. (D, E) Dissected whz hearts injected with KCl (D) and wild-type tbx20 mRNA (E) are stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize cardiomyocyte proliferation. (F) whz mutant hearts injected with wild-type tbx20 mRNA show significantly increased numbers of ventricular cardiomyocytes at 72 hpf (E) compared to control-injected whz mutants (D) (whz+tbx20 mRNA: 142.5±10 SD, whz + KCl: 87.82±10 SD, n = 10; p = 0.0001). (G) Ventricular cardiomyocyte proliferation in whz mutant embryos injected with tbx20 mRNA is significantly enhanced compared to control injected mutants at 72 hpf (whz+tbx20 mRNA: 7±3% SD, whz+KCl: 1±2% SD, n = 10; p = 0.0001).

Overexpression of tbx20 in wild-type zebrafish embryos results in reduced ventricular cardiomyocyte proliferation.

(A, B) Dissected wild-type hearts injected with KCl (A) and tbx20 mRNA (B) at 72 hpf, stained against MEF-2 (red) after incorporation of 5-ethynyl-2'-deoxyuridine (EdU; green) to visualize the effect of tbx20 overexpression on cardiomyocyte proliferation. (C) Wild-type zebrafish hearts injected with wild-type tbx20 mRNA show significantly reduced numbers of ventricular cardiomyocytes at 72 hpf compared to control injected hearts (wt+KCl: 148.6±2.9; wt + tbx20 mRNA: 64.0±2.017, n = 10, p< 0.0002). (D) Ventricular cardiomyocyte proliferation in wild-type embryos injected with tbx20 mRNA is significantly reduced compared to control injected embryos at 72 hpf (wt+KCl: 8.9 ± 0.38; wt+tbx20 mRNA: 1.1±0.28, n = 10, p = 0.0001).

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